Conjugated Polymer-Based Photoelectrochemical Cytosensor with

Jan 25, 2018 - In this work, a new photoelectrochemical (PEC) cytosensor was constructed by using cationic polyfluorene derivative, poly(9,9-bis(6′-...
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A Conjugated Polymer-Based Photoelectrochemical Cytosensor with Turn-On Enable Signal for Sensitive Cell Detection Shanshan Liu, Ping He, Sameer Hussain, Huan Lu, Xin Zhou, Fengting Lv, Libing Liu, Zhihui Dai, and Shu Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18275 • Publication Date (Web): 25 Jan 2018 Downloaded from http://pubs.acs.org on January 28, 2018

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A Conjugated Polymer-Based Photoelectrochemical Cytosensor with Turn-On Enable Signal for Sensitive Cell Detection Shanshan Liu,a,b Ping He,b Sameer Hussain,b Huan Lu,b Xin Zhou,b Fengting Lv,b Libing Liu,b Zhihui Dai,a* and Shu Wangb* a

School of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, P. R. China b

Beijing National Laboratory for Molecular Science, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China E-mail: Email: [email protected]; [email protected]

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ABSTRACT: In this work, a new photoelectrochemical (PEC) cytosensor was constructed by using cationic

polyfluorene

derivative,

poly

(9,9-bis(6’-N,N,N,-trimethylammonium)hexyl)fluorene-co-alt-1,4-phenylene) bromide) (PFP) as the photoelectric-responsive material for sensitive cell detection. Positive-charged PFP with high photoelectric conversion efficiency can generate robust photocurrent under the light illumination. In the PEC cytosensor, the 3-phosphonopropionic acid was linked to the ITO electrode followed by the modification with anti-EpCAM (epithelial-cell-adhesion-molecule) antibody via amide condensation reaction. Thus, target SKBR-3 cells with overexpressed EpCAM antigen could be captured onto the electrode via the specific antibody-antigen interactions. Upon adding cationic PFP, a favorable electrostatic interaction between cationic PFP and negatively charged cell membrane led to a turn-on detection signal for target SKBR-3 cells. This new cytosensor not only exhibits good sensitivity because of the good photoelectric performance of conjugated polymers, but also offers decent selectivity to target cells by taking advantage of the specific antibody-antigen recognition. KEYWORDS: photoelectrochemical cytosensor, conjugated polymers, turn-on signal, SKBR-3 cell, sensitive detection

1. INTRODUCTION Photoelectrochemical (PEC) measurement with light as excitation source and photocurrent as detection signal has attracted extensive attention as an emerging analytical technique in recent years.1-5 Owing to the remarkable merits including convenient operation, low background signal and high sensitivity,6-8 a variety of PEC biosensors have been developed for the detection of biomacromolecules,9-11 metal ions,12-14 chemical molecules15-16 and cells.17-18 Since, the photoelectric conversion efficiency of the photoelectric-responsive materials plays a crucial role in the performance of PEC biosensors, considerable efforts have been focused on the exploitation of good photoactive ACS Paragon Plus Environment

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semiconductor materials, including inorganic species (such as CdTe,19 TiO2,20-22 ZnO,9,23 PbS24 nanomaterials) and organic species (such as porphyin,25 phthalocyanine,26 ruthenium complexes27). However, inorganic photoelectric-responsive materials suffer from low photovoltaic conversion efficiency, strong photocorrosion, limited spectral absorption range or biotoxicity. while organic photoactive species possess inferior photostability or charge transfer performance.28-29 Although the combination of wide band-gap semiconductors with narrow band-gap ones or inorganic semiconductors with organic molecules could overcome some drawbacks, the selection for the energy level matching materials and operation system remains problematic.30 Therefore, it is still required to explore new photoelectric-responsive materials for developing high-performance PEC biosensors. Conjugated polymers (CPs) with π-conjugated backbones and charged side-chains possess robust light-harvesting ability, excellent photostability and good semi-conductive characteristic.31 These features endow them promising applications in photocatalytic water splitting,32 bioimaging33 and disease therapy.34 CPs could generate electron-hole pairs under light illumination. The electrons migrating along the backbone could be captured by electron accepter accompanied with the elimination of holes, resulting in the generation of stable photocurrent.35 However, the exploration of CPs as photoelectric-responsive materials in PEC biosensors is still rare. It should be noted that, in previous works, the developed cytosensors are usually dependent on turn-off signal to achieve the purpose of detecting cells, where the dielectric behavior of cells is generally utilize to block charge transfer and decrease the PEC response of photoelectric-responsive materials.5, 36-37 The current detection method based on more convenient PEC is cost-effective (unlike optical techniques) and require low operating potential. It is unlike electrical or electrochemical measurements which usually necessitate high operating voltage. Moreover, PEC technique entirely separates the detection signal (current) from excitation source (light) which in turn reduces the undesired background signal to deliver high sensitivity for bioanalysis compared with the classical methods such as fluorescence. In this work, for the first time we employed cationic CPs as the photoelectric-responsive material instead of traditional semiconductors to construct a PEC cytosensor with turn-on enable signal for sensitive cell detection. ACS Paragon Plus Environment

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2. RESULTS AND DISCUSSION The construction and working mechanism of PEC cytosensor is illustrated in Scheme 1. The positive-charged

polyfluorene

derivative,

poly(9,9-bis(6’-N,N,N,-trimethylammonium)hexyl)fluorene-co-alt-1,4-phenylene) bromide) (PFP), is chosen here as photoelectric-responsive material due to its high light adsorption coefficient and good semiconducting property. The ITO electrode was modified with 3-phosphonopropionic acid by the esterification reaction between hydroxyl groups on the surface of ITO and phosphate groups, which provides carboxyl groups for further antibody modification. The anti-epithelial-cell-adhesion-molecule (anti-EpCAM) antibody was then linked onto the ITO surface via amide condensation reaction. Subsequently, Bovine serum albumin (BSA) was coated onto the ITO surface to minimize the nonspecific binding of cells. Thus, target SKBR-3 cells with overexpressed EpCAM antigen can be captured onto the electrode via specific antibody-antigen interactions. Finally, the cationic PFP was added that can successfully bind with negatively charged cell membrane through electrostatic interactions. The loading of PFP can produce photocurrent under light irradiation to provide a turn-on response signal, hence, specific detection of SKBR-3 cells is realized using the PEC cytosensor.

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Scheme 1. Schematic illustration of construction and working mechanism of PEC cytosensor for target cell detection using cationic PFP as photoelectric-responsive material.

To

confirm

the

successful

linking

of

anti-EpCAM

antibody

onto

ITO

electrode,

ITO/BSA/anti-EpCAM and ITO/BSA were respectively treated with rabbit anti-mouse IgG labeled with Alexa Fluor 555 dye that could specifically recognize anti-EpCAM antibody.38 After incubating at 37 °C for 30 min, they were washed three times with PBS (pH = 7.4, 0.01 M), and then observed under fluorescence microscope. As shown in Figure S1A, obvious green fluorescence of Alexa Fluor 555 was observed for ITO/BSA/anti-EpCAM electrode, while neglectable fluorescence was observed for ITO/BSA electrode without anti-EpCAM antibody modification (Figure S1B). The surface density calculated for the surface-immobilized antibodies using BCA assay was found to be about 5.7 ng/mm2. This confirms the successful linking of anti-EpCAM antibody onto the ITO electrode. We then investigated the capture and detection ability of ITO/BSA/anti-EpCAM for EpCAM antigen overexpressed SKBR-3 cells. The fabricated PEC cytosensor was immersed in target cell suspension at a certain concentration and incubated at 37 °C for 1 h, followed by washing with PBS (pH 7.4, 0.01 M) to remove the nonspecifically adsorbed cells. Then, the cytosensor was immersed into PFP solution (50 ACS Paragon Plus Environment

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µM)

to

absorb

positive-charged

PFP

through

electrostatic

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interactions.

The

image

of

ITO/BSA/anti-EpCAM/BSA/cell/PFP was recorded by the fluorescence microscopy (Figure 1 and S2). The bright field image showed that cells were successfully captured on the surface of ITO/BSA/anti-EpCAM. Furthermore, the ITO electrode emitted bright fluorescence upon excitation at 405 nm, which suggested that positive-charged PFP could bind to the surface ITO electrode in virtue of SKBR-3 cells.

Figure 1. Fluorescence microscopy images of ITO/BSA/anti-EpCAM/cells/PFP electrode (1×104 cell·mL-1).

The photocurrent responses of ITO electrode with stepwise modifications were investigated in the PBS (0.1 M, pH = 7.4) containing ascorbic acid (AA) under white light irradiation at applied potentials. Before PEC response tests, the measurement conditions including the concentration of ascorbic acid and applied potential were optimized. The bias potential was a significant factor influencing the generation of the photocurrent (Figure S3A). The photocurrent increased slightly on changing the applied voltage from -0.4 V to -0.2 V and then deceased sharply with further changing it from -0.2 V to 0.2 V. Considering the low applied potential as beneficial for the elimination of interference from other species coexisting in the samples, -0.2 V was chosen as the optimal applied potential in the PEC measurements. In addition, the effect of AA concentration on the PEC response was also investigated (Figure S3B). The photocurrent intensity increased gradually when the concentration of AA changed from 0 M to 0.09 ACS Paragon Plus Environment

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M and reached a plateau at 0.09 M. Therefore, 0.09 M AA and applied potential of -0.2 V were selected as the optimized parameters for the PEC measurement in all experiments. As shown in Figure 2A, the bare ITO, ITO/anti-EpCAM, ITO/BSA/anti-EpCAM and ITO/BSA/anti-EpCAM/cells could not generate

photocurrents

(Figure

2A,

plots

a-d).

When

PFP

was

introduced

onto

the

ITO/BSA/anti-EpCAM/cells electrode, a robust cathode photocurrent was observed since the PFP could be excited and generated electron-hole pairs upon light illumination (Figure 2A, plot e).39 At the negatively applied bias of -0.2 V, the protons in the PBS containing AA consumed the electrons on the LUMO orbital and electrons from the ITO electrode transferred to scavenge photoinduced holes on the HOMO orbital. AA as a nontoxic electron donor could also neutralized a portion of photo-generated holes, which inhibited the electron–hole recombination.40 The efficient charge separation and transfer guaranteed the generation of robust cathode photocurrent. Meanwhile, the cathodic photocurrent was relatively stable periodically over time (Figure 2A, plot e). It was noted that the bare ITO electrode without antibody and cells could also generate weak photocurrent upon immersing it into PFP solution (50 µM) probably due to nonspecific absorption (Figure 2B, plots b-d). However, such photocurrents were much weaker than that of ITO/BSA/anti-EpCAM/cells/PFP electrode (Figure 2B, plot a), owing to the strong adsorption capacity of cell membrane to PFP by electrostatic interactions.

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Figure 2. (A) Photoelectrochemical responses of modified ITO electrodes: bare ITO (a), ITO/anti-EpCAM (b), ITO/BSA/anti-EpCAM (c), ITO/BSA/anti-EpCAM /cell (5×104 cell·mL-1) (d), ITO/BSA/anti-EpCAM/cell/PFP (e). (B) Photoelectrochemical responses of modified ITO electrodes: ITO/BSA/anti-EpCAM/cell/PFP (5×104cell·mL-1) (a), ITO/BSA/PFP (b), ITO/BSA/anti-EpCAM/PFP (c), ITO/BSA/cell/PFP (d). The measurement was performed in the PBS (0.1 M, pH = 7.4) containing 0.09 M ascorbic acid (AA) under white light irradiation at an applied potential of -0.2 V.

The PEC cytosensor was then applied to the quantitative determination of SKBR-3 cells. To accomplish this, different ITO/BSA/anti-EpCAM electrodes were dipped into SKBR-3 cells suspension of variable concentrations (0, 1 × 102, 5 × 102, 1× 103, 5 × 103, 1 × 104, 5 × 104, 1 × 105, 5 × 105, 1 × 106 cell·mL-1) followed by incubation at 37°C for 1 h and washing with PBS (pH 7.4, 0.01 M). During measurements, an increment in the intensity of photocurrent was observed with increasing the concentration of cells which was likely due to an enhanced PFP captured by modified electrode (Figure 3A). A good linear relationship can be obtained between the ∆I (∆I = I − I0; where I is the photocurrent ACS Paragon Plus Environment

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for the SKBR-3 cells at different concentrations and I0 is the background signal when SKBR-3 cell concentration was zero) and the logarithmic value of the SKBR-3 cell concentration in the range from 1.0×102 to 5.0×105 cell·mL-1 (Figure 3B). The linear regression equation was ΔI (µA) = -2.706 + 1.769 lgC (cells per mL) with a correlation coefficient of 0.996. The detection limit was estimated to be 24 cells per mL at a signal-to-noise ratio of 3. In addition, the parallel measurements of five independent electrodes incubated with cell suspension (1.0×104 cell mL-1) were recorded and the relative standard deviation (RSD) was calculated to be about 8.4%, which suggested that the constructed PEC cytosensor owned good reproducibility and reliability. These results revealed that our cytosensor possessed satisfactory analytical performance towards SKBR-3 cells. Compared with previous reports,41-43 the proposed PEC cytosensor platform exhibited better detection capability based on the excellent PEC property of conjugated polymers. Furthermore, most of the methods41,42 reported for selective capturing of target cells by specific surface-immobilized antibodies were based on classical “turn-off” signal mechanism which usually exhibited high background interference and limited range of linearity. It was unlike “turn-on” signal approach employed in our work which showed wide range of linearity.

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Figure 3. (A) PEC responses of the cytosensor to SKBR-3 cells with different concentrations at 0, 1 × 102, 5 × 102, 1× 103, 5 × 103, 1 × 104, 5 × 104, 1 × 105, 5 × 105, 1 × 106 cell·mL-1 (a-j). (B) Linear calibration curve between photocurrent change and the logarithmic value of the SKBR-3 cell concentration. The selectivity of PEC cytosensor towards cells was also investigated.

As shown in Figure 4, the

photocurrent against Jurkat or HeLa cells with negatively expressed EpCAM was negligible compared with that of SKBR-3 cells with overexpressed EpCAM, which indicated that the PEC cytosensor had the good capability for specific detection of SKBR-3 cells. We further applied the cytosensor to detect SKBR-3 cells in the Roswell park memorial institute-1640 (R-1640) medium and R-1640 with 10% fetal bovine serum (FBS) mixed medium, respectively (Figure S4). The results showed that the PEC cytosensor worked well in the medium containing complicated component samples, suggesting the potential for application in clinical diagnoses and prognosis of cancer.

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Figure 4. Photoelectrochemical responses of the cytosensor to SKBR-3, Jurkat and HeLa cells. The measurement was performed in the PBS (0.1 M, pH = 7.4) containing 0.09 M ascorbic acid (AA) under white light irradiation at an applied potential of -0.2 V. The cell concentration is 1 × 104 cell·mL-1.

3. CONCLUSION In this work, a new photoelectrochemical (PEC) cytosensor with turn-on signal was constructed by using cationic conjugated polymer as the photoelectric-responsive materials. The positive charged conjugated polymer could be absorbed via electrostatic interaction onto the ITO electrode modified with cells. Under white light irradiation, conjugated polymer formed electron-hole pairs resulting in the generation of stable photocurrent. The PEC cytosensor exhibited photocurrent variation after capturing different amounts of SKBR-3 cells and a wide linear relation between photocurrent change and cell concentration. This new cytosensor exhibited good sensitivity with a detection limit of 24 cells per mL because of the good photoelectric performance of conjugated polymers. Remarkable selectivity in the assay of SKBR-3 cells was realized by taking advantage of the specific antibody-antigen recognition. This work paved a simple method for the assay of cells, showing a promising perspective for the construction of versatile PEC platforms and application for cancer prognosis.

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Supporting Information.

Page 12 of 18

Experimental procedures, additional Figures S1-S4. This material is

available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] (Z. D.); [email protected] (S.W.)

AUTHOR CONTRIBUTIONS S. L. and X. Z. performed the photoelectrochemical experiments. P. H. and H. L. conducted the fluorescence imaging studies. S. H. performed the surface density experiment and grammatically improved the manuscript. S. L., F. L., L. L., Z. D. and S. W. designed the experiments and conceptualized the work. All authors discussed the results and wrote the manuscript.

ACKNOWLEDGEMENTS The authors are grateful to the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA09030306), the National Natural Science Foundation of China (Nos. 21661132006, 21625502, 21475062, 21533012, 21473221).

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Evolution. ACS Appl. Mater. Interfaces 2017, 9, 10355-10359. (36) Wu, P.; Pan, J.; Li, X.; Hou, X.; Xu, J.; Chen, H. Long-Lived Charge Carriers in Mn-Doped CdS Quantum Dots for Photoelectrochemical Cytosensing. Chem. Eur. J. 2015, 21, 5129-5135. (37) Zhao, X.; Zhou, S.; Jiang, L.; Hou, W.; Shen, Q.; Zhu, J. Graphene–CdS Nanocomposites: Facile One-Step Synthesis and Enhanced Photoelectrochemical Cytosensing. Chem. Eur. J. 2012, 18, 4974-4981. (38) Wen, C.; Wu, L.; Zhang, Z.; Liu, Y.; Wei, S.; Hu, J.; Tang, M.; Sun, E.-; Gong, Y.; Yu, J.; Pang, D. Quick-Response Magnetic Nanospheres for Rapid, Efficient Capture and Sensitive Detection of Circulating Tumor Cells. ACS NANO 2014, 8, 941-949. (39) Wu, Z.; Sun, C.; Dong, S.; Jiang, X.; Wu, S.; Wu, H.; Yip, H.; Huang, F.; Cao, Y. n‑Type Water/Alcohol-Soluble Naphthalene Diimide-Based Conjugated Polymers for High-Performance Polymer Solar Cells. J. Am. Chem. Soc. 2016, 138, 2004-2013. (40) Wang, F.; Wang, W.; Wang, X.; Wang, H.; Tung, C.; Wu, L. A Highly Efficient Photocatalytic System for Hydrogen Production by a Robust Hydrogenase Mimic in an Aqueous Solution. Angew. Chem. Int. Ed. 2011, 50, 3193-3197. (41) Liu, F.; Zhang, Y.; Yu, J.; Wang, S.; Ge, S.; Song, X. Application of ZnO/Graphene and S6 Aptamers for Sensitive Photoelectrochemical Detection of SKBR-3 Breast Cancer Cells Based on a Disposable Indium Tin Oxide Device. Biosens. Bioelectron. 2014, 51, 413-420. (42) Zhang, X.; Li, S.; Jin, X.; Li, X. Aptamer Based Photoelectrochemical Cytosensor with Layer-by-Layer Assembly of CdSe Semiconductor Nanoparticles as Photoelectrochemically Active Species. Biosens. Bioelectron. 2011, 26, 3674-3678. (43) Feng, L.; Chen, Y.; Ren, J.; Qu, X. A Graphene Functionalized Electrochemical Aptasensor for Selective Label-Free Detection of Cancer Cells. Biomaterials 2011, 32, 2930-2937. ACS Paragon Plus Environment

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